Heterotrimeric G proteins sit at the cytoplasmic face of membranes and transduce signals from cell surface receptors to intracellular effectors. The heterotrimer consists of the alpha subunit that binds GTP and the beta and gamma subunits that form a tight complex. Most of the G alpha subunits undergo palmitoylation, the reversible, post-translational addition of palmitate to an amino-terminal cysteine residue. Palmitoylation increases the membrane affinity of the G alpha subunit as well as modulating protein interactions with the beta/gamma subunits, receptors and effectors. Activation of G proteins by a receptor increases the palmitate turnover on the alpha subunit. The enzymes responsible for palmitoylation and depalmitoylation of proteins have been elusive. To better understand the regulation of this cycle, we are investigating the enzyme responsible for G-protein palmitoylation. We developed a strategy to purify protein fatty acyl transferase (PAT) activity from rat livers that took advantage of recent knowledge on the cellular location and inhibition of PAT activity. We determined that three different thiolases have PAT activity in the presence of imidazole and therefore started the purification with a plasma membrane fraction to minimize the contamination with these enzymes. After detergent extraction of the plasma membrane fraction, the PAT activity was enriched about 90 fold by sequential chromatography including affinity chromatography to a cerulenin-based inhibitor of palmitoylation. The partially purified PAT activity 1) was lost with treatments to degrade or denature proteins, 2) could acylate tubulin, Galpha i and RGS16 and 3) showed a preference for palmitate and to a lesser degree other long chain fatty acids. This purification procedure is a significant advance over previous efforts at PAT purification and a starting point for a proteomic approach for identification of mammalian protein fatty acyl transferases.